Co-pending patent application W092/05181 herein incorporated by reference discloses the purification of polynucleotides (particularly DNA) derived from human or animal cells by the removal of protein impurities. The proteins are removed by reaction with a cross-linked silica gel material having free CHO or CO groups, so as to remove the proteins as a solid or semi-solid material in a two-phase solvent system. The lower phase is chloroform and the upper aqueous phase contains the DNA. The protein-containing silica is present as an intermediate solid or semi-solid layer which is easily separated away. The cross-linked silica is preferably prepared by cross-linking silica gel with 3-aminopropyltriethoxysilane, followed by reaction with gluteraldehyde to introduce protein-reactive CHO groups.
However, DNA extracted from plants often contains polysaccharides as an impurity, which are not encountered with DNA derived from most other organisms. Such polysaccharides may have enzyme-inhibitory properties, which may subsequently prove troublesome should the DNA be subsequently subjected to enzyme digestion, for example restriction enzyme digestion. To date, most extraction methods have employed the expensive and time-consuming caesium chloride density gradient technique to remove these polysaccharides (Bendich A J, Anderson R S, Ward B L (1980) Plant DNA: long, pure and simple. In: Leaver Cj(ed) Genome Organisation and Expression, pp 31-33. New York: Plenum Press; Murray H G., Thompson W F.(1980) Nucleic Acids Res 8:4321-4325; Taylor B, Powell A (1982) Isolation of plant DNA and RNA. Focus 4:4-6.). Other methods have also been reported (Dellaporta S L, Wood J, Hicks J B (1983) Plant Mol Biol Rep 1:19-21. Zimmer E A, Newton K J (1982) A simple method for the isolation of high molecular weight DNA from individual maize seedlings and tissues. In: Sheridan W F (ed) Maize for Biological Research. Grand Forks, N.D.: University press, University of North Dakota.) however, such techniques are generally only applicable to a limited number of plant species and tissue types. The separation of polysaccharides from nucleic acids by the differential solubilities in the presence of cetyltrimethylammonium bromide has also been reported (Rogers S O, Bendich A J (1988) Plant Mol Biol Manu.A6:1-10.); but this procedure requires organic extraction which may cause some degradation of the DNA.
It is an object of the present invention to provide a convenient process for the isolation of polynucleotides from polysaccharide-containing mixtures.
This and other objects of the present invention will become apparent from the following description and examples.